OBSERVATION AND RESULTS

Delse P. Sebastian “Micropropagation of some important medicinal plantsof Kerala” Thesis. Department of Botany , University of Calicut, 2002

OBSERVATION AND RESULTS

Present investigations developed efficient and reproducible protocols for

the micropropagation of two important medicinal plants viz., Heliofrvpium

keralensis and Naregamia alafa through direct shoot induction, callus

regeneration, somatic embryogenesis and synseeds from various explants. The

observations and results of the experiments are entitled as follows

HEUOTROPIUM KERAL ENSIS

Efficient and reproductive protocols have been developed for

organogenesis and somatic embryogenesis of H. keralensis by culturing

explants such as shoot tip, node, internode, leaf and root on different media

supplemented with various hormonal combinations.

Among the three surface sterilants (sodium hypochlorite, ethanol and

mercuric chloride) tested, surface sterilization with 0.1% HgCI2 for 6 and 7 min

was most effective for explants of shoot system (shoot tip, node, internode and

leaf) and root respectively.

The problem of phenolic exudation was tried to control by using

additives such as 'activated charcoal (0.06-l%), PVP (1 g/l) and ascorbic acid

(l00 mg/l) or by periodic subculturing to the medium of same composition.

Among these weekly subculturing to the medium of same composition

produced best results. Addition of 1% activated charcoal was also an effective

method to overcome the problem. But the addition retarded growth in culture,

hence it was not used in subsequent experiments.

DIRECT SHOOT REGENERATION

For the induction of direct multiple shoots, different explants like Shoot

tip, node, and root were cultured on different media such as MS, B5 and White's

supplemented with different combinations and concentrations of growth

regulators.

i) Multiple shoot regeneration from shoot tip

Three different basal media (MS, B5, White's) were tested for selecting an

appropriate culture medium for Hkeralensis. Shoot tips showed elongation upto

2 cm. within 20 days when cultured on MS basal medium. However, shoot tips

cultured on B5 basal medium required 40 days for elongation up to 2 cm. and

shoot tips cultured on White's basal medium did not show any response even

after 50 days of culture. On the basis of this observation MS medium was

selected for further experiments.

Multiple shoots were formed from the explants when cultured on MS

medium supplemented with BA (0.5-4.0 mg/l) or BA (0.5-3.0 mg/l) + Kn (0.5-

3.0mg/l) or BA (0.5-3.0mgll) + Kn (0.5-3.0mgll) +IAA (0.5 mg/l) (Plate 1

A,B,C) (Table 12 ) . Of the two cytokinins (BA and Kn) tested, BA was effective

Table 12. Effect of growth regulators on shoot induction from shoot tip

explants of H. Keralensis

Growth regulator (mgll) BA

Shoot Length

(cm)

1 .O 1.5 2.0 3 .O

4.0

Kn 0.5

1 .O BA + Kn 0.1 0.1

0.5 0.5

1.0 1.0

2.0 2.0 3.0 3.0

4.0 4.0 BA+Kn+IA

2.0 2.0 0.5

3.0 3.0 0.5

BA+Kn+NA 0.5 0.5 0.5

1.0 1.0 0.5 bc- Basal callusing

% of Response No. of ShootsIExplant

Data fiom 20 replicates in two experirnents(Mean S E ) Growth period 50 days

70 75 8 5

85

70

30

40

35

70

80

90 90

85

80 85

100%

100%

6.2 2 0.25 8.47 t 0.73 10.31 20.11

13.14 + 0.33 12.7 t 0.16

1

1

1

4.68 2 0.39 7.8 2 0.13 12.1 2 0.62 14.2 2 0.57 10.3 + 0.18

10.1 f. 0.74

12 2 0.26

bc

bc

for the induction of multiple shoots. BA at different levels (0.5-4.0 mg/l) on MS

medium produced multiple shoots from the explants within 20 days. There was

a linear correlation between the increase in BA concentration upto the optimal

level and shoot multiplication. For direct shoot regeneration, 3.0 mg/l was the

optimum concentration of BA. At lower concentration (0.1 mg/l) of BA shoot tip

only elongated with an average length of 5 cm within 50 days (Table 12).

Addition of Kn along with BA on MS medium showed an enhancement in

shoot multiplication. A combination of ' BA (3.0 mg/l) + Kn (3.0 mg/l) on MS

medium produced highest number (14) of shoots from the explants (Plate 1B).

Auxins (IAA and NAA) when used alone on MS medium no shoot

multiplication was observed from the explant. The combinations of auxins with

cytokinins (BA + Kn) on MS medium were less effective when compared to MS

medium supplemented with cytokinins (BA + Kn). However the auxin cytokinin

combination promoted elongation of shoots. The average shoot length was

highest (6.0 cm) on MS medium supplemented with BA (2 mg/l) + Kn (2 mg/l)

+ IAA (0.5 mg/l) (Table 12).

ii) Multiple shoot regeneration from nodal explants

Multiple shoot regeneration from nodal explants was observed on MS

medium supplemented with BA (0.5-4 mg/l) or BA (0.5-4.0 mg/l) + Kn (0.5-4.0

mg/l) or BA (0.5-4.0mg/1) + Kn (0.5-4.0 mg/l )+ IAA (0.5 mg/l) (Plate 2 B&).

The explants cultured on these media showed their first response by initial

enlargement of the existing axillary buds followed by bud break within two

weeks. BA at the range 0.5- 4.0 mg/l induced multiple shoots from the explant

within 20 days. Number of shootsjexplant increased with increase in BA

concentration upto 3.0 mg/l. Further increase in concentration of BA decreased

shoot multiplication from the explant. However length of shoots decreased

with increase in BA concentration. Kn (0.5-3.0 mg/l) when supplemented

singly on MS medium no multiple shoot formation was observed. However,

single shoot was formed from each axil of the nodal explants at 1.0 mg/l and

2.0 mg/l concentrations of Kn in MS medium (Plate 2 A).

Combination of Kn (0.5 - 3.0 mg/l) and BA (0.5 - 3.0 mg/l) in MS

medium showed high rate of shoot multiplication from the nodal explant, about

12 shoots were formed within three weeks. Highest number of shoots were

induced on the MS medium containing 3mg/l BA and 3mg/l Kn.

Addition of auxin (MA) in conjunction with cytokinins (BA + Kn) showed

negative effect on shoot multiplication. However, the combination was

favourable for shoot elongation (as in the case of shoot tip explants) (Table

13).

Table 13. Effect of growth regulators on shoot induction from nodal

explants of H. keralensis

Growth regulator (mg/l) BA

bc + 2.2 bc + 3.48

SE)

% of Response No. of 1 ShwWnode

0.5 0.5 0.5

1.0 1.0 0.5

100

100

Growth period 50 days

Shoot Length

(cm)

Data from 20 replicate3 in two experiments@kan

!

Callusing only Callusing only

Table 14. Effect of growth regulators on direct shoot induction from root explants of H. keralensis

Callusing only

Callusing only

Callusing only

Shoot Length

(cm)

Data from 20 replicates in two experiments(Mean S E ) Growth period 50 days

No. of Shoots/node

Growth regulator (mgll)

BA

%' of Response

iii) Multiple shoot regeneration from root explants

Direct shoot regeneration from root explants of H. keralensis was

observed on MS medium supplemented with BA (2-5 mg/l) and on MS medium

supplemented with BA (2-5 mg/l) and Kn (2-5 mg/l (Plate 3 A,B,C) (Table 14).

BA was found as the essential cytokinin for direct shoot regeneration from root

explants. The optimal concentration was 4.0 mg/l BA in MS medium for direct

shoot regeneration from root explants. Kn did not evoke any response when

tested alone with MS medium. However a combination of BA (4.0 mg/l) and Kn

(4.0 mg/l) was most effective for direct shoot regeneration from root explants

(Table 14 ).

All regenerated shoots were free from any basal callus formation at their

proximal end. Addition of IAA (0.5 mg/l) was found to be least effective either

singly or along with BA and Kn. Instead the combination induced callusing

from root explants.

iv) Effect of nutrient media

Mineral salt composition of culture media significantly affected direct shoot

regeneration of H. keralensis. To select the best medium for direct shoot

Figure 1. Effect of nutrient media on shoot induction from shoot tips of H. keralensis

l

~ Media ! I 1 / % of response

l media and % of response I

Figure 2. Effect of sucrose on shoot induction from shoot tips of

H. keralensis (MS medium with 3mgll Kn + 3mgll BA)

2 3 4

% of sucrose

l

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$$$$g $$$$g iiifilififi;

l

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regeneration, shoot tip explants were cultured on different nutrient media (MS,

Bs, White's) supplemented with BA (3mg/l) and Kn (3mg/l). Establishment rate

(90%) and multiplication (14) were highest on MS medium (Figure l ) .

v) Effect of sucrose concentration

To find out the optimal level of sucrose for the induction of multiple

shoots of H. keralensis, shoot tip explants were cultured on MS medium with

varying levels of sucrose (l-5'/0) along with 3.0 mg/l BA + 3.0 mg/l Kn. Shoot

multiplication was observed with high degree of variation in all the

concentrations of sucrose tested and found that 3% was the optimum.

Increase or decrease in sucrose concentration resulted in a decrease in shoot

multiplication (Figure 2).

vi) Subculturing

To study effect of subculturing on shoot multiplication, shoot tips were

excised from li7 vitro shoots and cultured on MS medium with BA (3.0 mg /I)

and Kn (3.0 mg /I). No significant change in shoot multiplication was observed

during subculture.

INDIRECT SHOOT REGENERATION

The shoot regeneration potential through callus phase from various

explants viz. node, internode, leaf and root to various concentrations and

combination of growth regulators in MS medium was studied.

i) Callus induction

For callus induction the explants were cultured on MS medium

supplemented with BA, Kn, 2iP, IAA, NAA and 2,4-D either alone on in

combinations (Table 15).

Effect of BA

Callus formation was observed on MS medium containing BA at the

range 2- 4 mg/l from leaf and intemodal (Plate 4 A) explants. These calli were

green, friable and were meristematic in nature. However, there was a

progressive increase in the amount of callus with the increase in BA

concentration upto 3 mg/l. Further increase in BA concentration beyond the

optimal level (3 mg/l) did not show any progressive change in callus

proliferation. Root and node explants did not produce any callusing on MS

medium supplemented with BA (Table 15).

Effect of Kn

MS medium with Kn (0.5 - 3.0 mg/l) was found as ineffective in inducing

callus from any explant tried (Table 15).

Table 15. Effect of growth regulators on callus induction from various explants of H. keralensis

Effect of IAA

MS medium supplemented with IAA at the range 0.5 - 4.0 mg/l induced

callus from leaf, internode and nodal explants within 12 days. Higher

concentration of IAA (1 3mg/l) was effective for callus induction from root

explants. The calli were pale white and friable (Plate 5 B). The calli later

turned brown and died within 60 days.

Effect of 2,4-D

MS medium fortified with 2,4-D (0.5 - 3.0 mg/l) was found effective in

inducing callus from leaf, internode and node explants within 10 days. The calli

were pale white and friable. The calli later turned brown and died within 50

days(Plate 48). Root explants also produced calli at higher concentration of

2,4-D (2 2 mgjl).

Effect of 2,4-D + BA

Various explants were cultured to study the synergistic effect of growth

regulators 2,4-D (0.5 -l.Omg/l) and BA (0.5 - 2.0mg/l) in callus induction of H,

kernlensis. Among the tested concentrations, l.Omg/l 2,4-D + 2.0mg/l BA

were most effective and produced enormous amount of calli from all the

explants cultured. These calli were pale green, friable and meristematic (Plate

5 A). The calli regenerated shoots up on culture on shoot induction medium.

Effect of 2,4-D + 2iP

MS medium containing the combinations of 2,4-D + 2iP was found best

for callus induction of H. keralensis. Callus proliferation was also high in this

combination. Callus induction was observed within 10 days from leaf, internode

and node explants. Calli developed on root explants only after 15 days. The

calli were green, friable (Plate 6 A) and had the potential to regenerate shoots

on shoot induction medium. Among the tested concentrations, 0.5 mgjl 2,4-D

+ 3.0 mgjl 2 iP was found as optimal for callus induction and proliferation from

all the explants.

ii) Callus regeneration

An enormous amount of calli were obtained from nodal explants on MS

medium supplemented with 2,4-D (0.5 mg/l) + 2 iP (3.0 mgjl). The same

callus was selected for regeneration studies to find out optimum growth

regulator combination on MS medium for callus regeneration. A combination of

BA (3.0 mg/l) + IAA (0.5 mg/l) on MS medium produced highest number of

shoots (11) per gram calli (Plate 5 D). Of the two cytokinins (BA and Kn)

tested, BA effectively regenerated shoot from calli (Plate 4 C) however, Kn with

MS medium was ineffective for the regeneration of shoots from the calli (Table

16).

Table 16. Effect of growth regulators on callus regeneration of H. keralensis

Data fiom 20 replicates in two experiments (Mean &SE) Growth period 50 days

No. of shoots/Grarn of Callus

3.7 + 0.76 4.4 + 0.28 7.6 + 0.42 9.8 + 0.17 9.4 + 0.24

- - -

-

2.7 + 0.41 5.2 + 0.12 7.8 5 0.57

10.8 + 0.32 10.6 + 0.63

Growth regulator(mg/l)

BA

0.5

1.0

2.0

3 .O

4.0

Kn

0.5

1 .O

2.0

3 .O

BA + IAA 0.5 0.5

1.0 0.5

2.0 0.5

3.0 0.5

4.0 0.5

% Response

60

7 5

75 80

80

Proliferation only

Proliferation only

Proliferation only

Proliferation only

40

65

60

7 5

7 5

Though callus induction was observed with various growth regulators on

MS medium, only calli obtained on MS medium containing BA, BA + 2,4-D &

2,4-D + 2 ip were found to be regenerative on subsequent cultures. The regeneration capacity of various calli were tested by subculturing on MS

medium supplemented with LAA (0.5 mg/l) and BA (3.0 mg/l) (Plates 4 CID; 5

D; 6 B). Weekly transfer of callus to medium with the same composition was

necessary to retain morphogenic potential of the calli. Brown coloured calli

formed from various explants or light green calli which turned brown in the

absence of subculturing at weekly intervals never showed regeneration (Plate 5

C). However in the shoot regenerated cultures also the part of the calli which

did not regenerate shoots within 30 days turned brown but it did not affect

growth of regenerated shoots.

SOMATIC EMBRYOGENESIS

Somatic embryogenesis in H. keralensis was established by the

subculturing of embryogenic callus on MS hormone free medium.

i) Induction of embryogenic calli

Leaf, internode and nodal explants were cultured on MS medium fortified

with 2,4-D, IAA, BA and 2iP either alone or in combinations to induce

embryogenic calli (Table 17).

Table 17. Effect of growth regulators on embryogenic calli induction from leaf, node and internodal explants of H. keralensis

Data fiom 20 replicates in two experiments Growth period 90 days E-Embryogenic calli NE-Non embryogenic calli

No. of embryos/g of

calli in suspension

- -

-

- - - -

5.1

6.2

11.8

8.7

- - -

- -

Growth regulators

(mg/l)

IAA

1 .O

2.0

2,4-D 1 .O 2.0

IAA+BA

0.5 1.0

0.5 2.0

0.5 3.0

0.5 4.0

IAA+2iP 0.5 1.0

0.5 2.0

0.5 3.0

0.5 4.0

2,4-D + 2iP 0.5 0.5

0.5 1.0

0.5 2.0

2,4-D + BA 0.5 1.0

0.5 2.0

Embryogenic Potential

NE NE

NE NE

NE NE NE NE

E E

E

E

NE NE

NE

NE NE

% of Response

Leaf

85

90

85

90

85

80

90

85

5 0

65

60

70

65

80

85

65

70

Internode

70

95

90

80

70

8 5

80

90

45

50

65

60

85

85

90

70

7 5

Node

80

95

100

90

7 5

90 90

8 5

40

5 5

70

70

70

85

85

80

80

43

All explants responded well and callus induction was observed within 10

days in all the tested combinations. Nodal explants responded earlier (within 7

days) followed by internode (8 days) and leaf ( l 0 days) explants. However,

later experiments revealed that only the calli formed on IAA (0.5 mg/l) and 2

iP (1- 4 mgjl) combinations in MS medium were embryogenic. Other calli were

non-embryogenic. The embryogenic calli were white and non-friable (Plate 7

B). But non-embryogenic calli were white and friable (Plate 7 A). Combination

of IAA (0.5mgJl) + 2 iP (3.0 mgjl) on MS medium produced calli with highest

embryogenic potential from all the explants tested.

ii) Induction and maturation of somatic embryos

Embryogenic calli upon transfer to MS hormone free suspension medium

showed proliferation and formation of somatic embryos. Somatic embryos of

various developmental stages (Globular, heart, torpedo) (Plate 7D,E) were

formed from embryogenic calli within 90 days. Weekly transfer of

embryogenic calli to fresh liquid medium of same composition was an essential

requisite for the formation of somatic embryos. Failure in subcc~lture resulted in

browning and subsequent death of calli. Calli from nodal explants on MS + IAA (0.5mgJl) + 2 iP (1-4 mgjl) combinations produced globular embryos even

in semi-solid MS hormone free medium (plate 7C). However, the embryos

turned brown soon after their formation, but browning did not affect the

germination capacity of somatic embryos.

Figure 3.Effect of sucrose on somatic embryogenesis of H.

keralensis

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% of sucrose

In general the somatic embryos passed through the normal

developmental stages to root and shoot differentiation when cultured on MS

semisolid medium with or without growth regulators. Frequency of germination

was 55% on basal MS medium. Higher frequency (65%) of germination was

obtained on MS medium supplemented with 0.5 mg/l Kn. (Plate 7 FIG)

iii) Effect of sucrose

Effect of sucrose concentration on somatic embryogenesis was studied

by incorporating varying levels of sucrose (1-5 %) on embryogenic .calli

inducing medium (MS medium supplemented with 0.5 mg/l 2,4-D and 3.0 mg/l

2 iP.). Nodal explants cultured on the media produced calli within 20 days. The

calli were subcultured on MS hormone free liquid medium to find out the

number of somatic embryos formed per gram of calli. . The level of sucrose

significantly affected em bryogenesis. Number of somatic embryos per gram of

calli increased with an increase in sucrose concentration upto 4 g/l in induction

medium (Figure 3). Further increase of sucrose concentration beyond 4% in

induction medium showed a decline in number of somatic embryos formed per

gram of calli on subsequent cultures.

SYNSEEDS

Among the various levels of sodium alginate (l, 2, 3, 4 and 5%) tested

for encapsulation 3% was the most suitable for encapsulation. At lower

concentrations (l-2%) the beads were delicate and were difficult to handle. At

higher concentrations (4-5%) beads were too hard which adversely affected

germination or development by preventing emergence of root and shoot. The

concentration of CaC12(2H20) solution also affected the formation and

germination/development of synseeds. Among the different concentrations

tested (25, 50, 75 and l00 mM) 50 mM was best for the formation and

subsequent germination/development of synseeds. The encapsulated embryos

using 3% sodium alginate and 50 mM CaC12(2H20) (Plate 8 B) showed signs of

germination within 20 days on hormone free MS medium (Plate 8 D). However,

the shoot buds encapsulated using the same concentrations of sodium alginate

and CaCI2 (2H20) (Plate 8 A) did not show any sign of growth when cultured on

same medium. But when cultured on MS medium containing 2.0 mgjl BA the

encapsulated shoot buds produced an average of 4 shoots (Plate 8 C) within 30

days. Storage of encapsulated embryos/shoot buds for 3-6 months at 4°C did

not affect their germination/development.

ROOTING I N VITRO

For root induction individual shoots (>3 cm) were excised and cultured

on MS medium supplemented with IAA (0.1 - 3.0mg/l) or IBA (0.1 - 3.0mg/l)

or NAA (0.1 - 3.0mgJl). Roots were formed on all the combinations tested

(with varying frequency) (Plates 10; 20; 30; 4E; 5E; 6C; 8E; 9A) (Table 18).

Higher concentrations of auxins (IAA > 2.0 mg/l, IBA > l mgjl; NAA > 0.5 mgjl)

Table 18. Effect of auxins (LAA, IBA & NAA) on in vifro rooting of H. keralensis

Data from 20 replicates in two experiments (Mean &SE) Growth period 50 days

No. of Roots

6.3 + 0.88 9.8 + 0.18 14.6 + 0.42 16.4 2 0.16

9.9+C+O.l1

7.2 + C 2 0.27

11.8 + 0.56 16.7 2 0.12 22.1 + 0.22

20.4 2 C + 0.63 16.8 + C + 0.47 13.2 + C + 0.29

4.1 k0.15

6.6 + 0.74 4.3 +C+0.19

5.2 + C + 0.37 3.7 + C f . 0.61

2.9 + C + 0.23

Growth regulators(mg/l) IAA

0.1

0.2

0.5

1 .O 2.0

3 .O

IBA 0.1

0.2

0.5

1 .O

2.0

3 .O

NAA

0.1

0.2

0.5

1 .O

2.0

3 .O

% Response

60

75

80

80

70

75

40 65

90

90

8 5 90

90

8 5

80

80

85.

75

caused basal callusing and subsequently decreased number of roots/shoot

(Table 18): IBA at 0.5 mg/ in MS medium was found as the most effective

combination for rooting of H. kenlensis shoots and produced highest number

of roots (25jshoot) with highest frequency (9O0/0) (Table 18).

SECONDARY METABOLITE ANALYSIS

Thin layer chromatography (TLC) performed the comparative analysis of

the alkaloids present in the roots of in vitro and field grown plants

Root extracts from in vitro plants showed 5 compounds (spots) with Rf

values 0.025, 0.056, 0.156, 0.263and 0.95. However root extracts from in vivo

plants showed only three compounds (spots) with Rf values 0.025, 0.056, and

0.956. This indicated that two more compounds were additionally present in

the roots of in vibogrown plants (Plate 10).

ACCLIMATIZATION AND TRANSFER TO FIELD CONDITIONS

Plantlets with fully expanded leaves and well-developed roots were

successfully hardened in the controlled conditions for 20days and eventually

established in natural soil. The plantlets with healthy root and shoot system

regenerated through various in vitro techniques were transferred after washing

with distilled water to small plastic pots containing sterile sand. The pots were

then covered with polythene bag/glass bottles (Plate 9 B) to maintain high

humidity. The plantlets were watered daily. The polythene bagslglass bottles

were removed after 20 days. Plantlets produced new leaves within 10 days

after acclimatization (Plates 1E; 2E; 3E; 4F; 5F; 6D; 7H 8F;! 9C) then they were

transferred to large pots containing sand and soil (1:l ratio). The potted plants

produced new leaves and showed healthy growth (Plate 9 D). Morphologically

there was no detectable variation between in vitro raised and naturally grown

plants. The plantlets developed via direct or indirect shoot regeneration showed

high percentage (80%) of survival while that by somatic embryogenesis

showed 65% survival only.

BIOCHEMICAL STUDIES

Biochemical analysis of primary calli (induced on MS medium supplemented

with IM 2mg/l) and regenerating calli (on MS medium supplemented with IAA

0.5 mg/l + BA 2 mg/l) showed that there were marked differences between the

two calli in terms of biochemical characteristics. In the regenerating callus

cultures of H.keralensi3 metabolites like sugars, proteins and phenolics were

high compared to primary callus. Peroxidase activity was also high in

regenerating calli (Table 19). The results from the analysis of polypeptide

patterns showed more types of polypeptides in the regenerating callus then in

the primary callus (Figure 4).

Table 19. Changes in metaboliie levels and a- of primary and regenerating callus cultures of H. kceralem& ,

PLATE 1. Micropropagation from shoot tip of kera/ensis.

A. Shoots induced on MS + BA (0.5mg/l)+Kn B. Shoots induced on MS + BA (2mg/l)+Kn (2mg/l) C. Elongated shoots on MS+ BA (0.5mg/1)(40 days old)

D. Roots induced on MS + IAA (0.5 mg/l) E. Hardened plantlet in a small pot

PLATE 2. Micropropagation from nodes of M keraJensis.

A. Shoots induced on MS + IAA (0.5 mg/l) B. Shoots induced on MS + BA (1.5 mg/i) C. Shoots induced on MS + BA (0.5 mg/l)+Kn (3mg/l) D. Roots induced on MS + IAA (1.0 mg/l) E. Hardened plantlet in a small pot

PLATE 3. Micropropagation from roots of H. kedensis

A. Shoots induced on MS+BA (2mg/l)

B. Shoots induced on MS+BA (4mg/l)+Kn (4mg/l)

C. Elongated shoots (after 40 days of growth)

D. Roots induced on MS+IAA (O.Smg/l)

E. Hardened plantlet in small pot

miL PLATE 3

PLATE 4. Micropropagation through indirect shoot r e g e m o n from internodes of H.kera/ensis

A. Green friable callus induced on MS+BA (3 mg/l) (20 days

old).

B. Brown coloured pale white callus induced on MS+2,4-D

(O.Smg/l) (40days old)

C. Indirect shoot regeneration on MS+BA (3mg/l)

D. Shoot regeneration on MS+BA (2mg/l)+IAA (O.Smg/l)

E. Roots induced on MS+ NAA (2mg/l)

F. Hardened plantlet in small pot.

PLATE 5. Micropropagation through indirect shoot regeneration from leaves and nodes of H. kera/ensis.

A. Green callus induced from leaf on MS+2,4-D (0.5mg/l) +BA

(lmg/l> (1Odays)

B. White friable callus induced from node on MS+2,4-D

(2mgIl)

C. Brown coloured white friable callus induced from node on

MS+2,4-D(3mgjl) (50 days old).

D. Shoot regeneration from callus on MS+BA (3mg/l) + IAA (0*5mg/l)

E. Roots induced on MS+IAA (0.5mg/l)

F. Hardened plantlet in small pot.

PLATE 6. Micropropagation through indirect shoot regeneration

from roots of H. keralensis

A. Callus induced on MS+2,4-D (0.5 mg/l)+ 2,iP (2mg/l)

(1Odays old).

B. Shoot regeneration from callus on MS +BA (3mg/l)

C. Roots induced on MS+IBA (0.2mg/l)

D. Hardened plantlet in small pot.

W= PLATE 6

PLATE 7. Plant regeneration via. Somatic embryogenesis of H. kera/ensis.

A. Non embryogenic callus induced on MS+IAA (0.5mg/l)

B. Embryogenic callus induced on MS+IAA (0.5mg/l)

+2iP(2mg/l).

C. Globular embryos induced from embryonic callus from nodal

explants on MS basal medium

D. Somatic embryoids showing different stages of development.

E. Mature somatic embryo (Torpedo stage)

F. Somatic embryo showing germination (l0 days).

G. Somatic embryo germinated plantlet.

H. Hardened plantlet in small pot.

PLATE 8. Encapsulation and germination of somatic embryosfshoot

buds of H. kerafensis.

A. Encapsulated shoot tips

B. Encapsulated somatic embryos

C. Multiple shoots developed from encapsulated shoot bud on

MS+BA (2mg/l) (25 days old.)

D. Germination of encapsulated somatic embryos on MS basal

medium.

E. Plantlet developed from synseed.

F. Hardened plantlet in small pot.

PLATE 9. Steps in hardening and field transfer of H, kem/ensis.

A. Plantlet with well developed shoot and root system.

B. Plantlet under hardening

C. Hardened plantlet in small pot.

D. Hardened plant under field conditions in pot.

PLATE 10. TLC analysis (for secondary metabolites ) of roots from in v i m and field grown plants.

I .. L b . . - . -A

PLATE 10 1

NAREGAMIA ALATA

For establishing an efficient protocol for micropropagation of N. alata,

various explants such as shoot tips, nodes, intemodes, leaves, petals and roots

of naturally grown plants were cultured ,on different media fortified with

different combinations and concentrations of growth regulators. The

experiments resulted inefficient and reproducible protocols for

micropropagation through organogenesis and somatic embryogenesis of this

important medicinal plant.

Among the different concentrations of various surface sterilizing agents

(sodium hypochlorite, ethanol and mercuric chloride) tested, 0.1%, HgClz

treatment for 10 min. produced best results for explants of shoot system (shoot

tip, leaf, node, internode and petal) and 0.1% HgClz treatment for 15 min. gave

best results for root explants.

DIRECT SHOOT REGENERATION

Attempts were made for establishing direct shoot regeneration with

different media augmented with different concentrations and combinations of

growth regulators. In the present studies direct shoot regeneration was

obtained from shoot tip, node and leaf explants of N. alafa.

i) Multiple shoot formation from shoot tip explants

Three different basal media (MS, B5, White's) were tested for selecting

an appropriate culture medium for N. alata. Shoot tips showed elongation

(60%) within 10 days on MS basal medium. Shoot tips cultured on Bs and

White's basal media did not show any response even after 20 days. On the

basis of the above observation MS medium was selected for further

experiments.

Multiple shoots were formed directly from shoot tip explant on MS

medium supplemented with varying combinations and concentrations of growth

regulators. (Table 20)

Effect of BA

Murashige and Skoog's medium containing BA (0.5 - 3.0 mg/l) was

found effective in producing multiple shoots from shoot tips. BA at 2 mg/l was

found as the optimal level in which shoot multiplication was high. Further

increase in BA (>2mg/l) concentration resulted in reduction in the number of

shoots (Table 20).

Effect of Kn

Murashige and Skoog's medium containing Kn (0.5 - 2.0 mg/l) was

ineffective in inducing multiple shoots from shoot tip explants of N. alata.

However, shoot tips elongated on Kn supplemented MS medium (Table 20).

Table 20. Effect of growth regulation on shoot induction from shoot tip explants on N. alata

Data from 20 replicates in two experiments (Mean H E ) Growth period 50 days SS= Single shoot

Growth regulators (mg/l)

BA

0.5

l .O

2.0

3 .O

Kn 0.5

1 .O

2.0

NAA + BA 0.5 0.5

0.5 1.0

0.5 2.0

0.5 3.0

0.5 4.0

IAA + BA 0.5 0.5

0.5 1.0

0.5 2.0

0.5 3.0

Shoot length

4.43 2 0.22 4.43 2 0.18 4.1 2 0.07

4 20.1

3.1 + 0.15 3.6 2 0.18 3.8 2 0.1

4.8 + 0.1 4.82 2 0.1 4.55 2 0.13 4.4 50.12

4.12 + 0.26

4.2 2 0.26 3.9 2 0.18 3.83 5 0.47 3.82 2 0.62

No. of shoots1 culture

9.3 2 0.76 12.3 2 0.45 2 1.9 + 0.57 21.1 2 0.63

SS elongated

SS elongated

SS elongated

9.9 + 0.49 13.6 2 0.39 21.2 + 0.57 27.4 2 0.52 25.2 2 0.3 1

10.2 2 0.14 13.6 2 0.72 18.0 2 0.32 17.4 5 0.51

-

% of Response

30

60

65

60

20

40

45

70

75

80

8 5

85

70

75

7 5 80

Effect of NAA + BA

Murashige and Skoog's medium supplemented with NAA (0.5 mg/l) and

BA (0.5 - 4.0 mg/l) was found as the most effective combination for shoot

multiplication of N. alata (Plate l1 A,B). Shoot induction was observed within

15 days in all the combinations tested. The optimal concentration of both NAA

(0.5 mg/l) and BA (3.0 mg/l) produced highest number (27) of shoots (Plate

11A). At higher concentrations of BA (> 3.0 mg/l) both shoot multiplication as

well as shoot elongation were declined (Table 20 ).

Effect of IAA + BA

Murashige and Skoog's medium fortified with IAA (0.5 mg/l) and BA

(0.5-3.0 mg/l) combination also produced multiple shoots from shoot tip

explants. However, the combination was less effective when compared to

NAA + BA combination in MS medium for shoot induction as well as shoot

elongation. At the optimal concentrations IAA (0.5mgJl) + BA (2.0 mg/l)

combination produced an average of 18 shoots per shoot tip.

ii) Direct shoot regeneration from nodal explants

Murashige and Skoog's medium augmented with growth regulators (BA,

Kn, IAA & NAA) either singly or in combinations was used to induce multiple

shoots from nodal explants of N. ahta (Table 21).

Table 21. Effect of Growth Regulators on Shoot Induction from Nodal Explants on N. alata

Data fiom 20 replicates in two experiments (Mean S E ) Growth period 50 days SS-Single shoot C- Callusing

% of Response

45

60 70

75

50

45

70

70 80

85 85 85 8 5

75 75

80 75

I

Shoot length

5.3 + 0.16 4.5 + 0.25 4.33 + 0.18 3.86 + 0.1 l

3.45 + 0.21 3.6 + 0.25

6.27 + 0.2 5.5 + 0.27 5.13 + 0.1 4.22 + 0.13 3.96 + 0. l 3.92 + 0.21 3.89 + 0.22

5.5 + 0.2 5.2 + 0.08

4.53 + 0.12 4.54 + 0.36

Growth regulators

(mg/l) BA

0.5

1 .O 1.5 3.0

Kn(mg/l) 0.5

1 .O 2.0

NAA + BA 0.5 0.5

0.5 1.0

0.5 + 1.5 0.5 2.0

0.5 3.0 0.5 4.0 1 .O 3 .O

IAA + BA 0.5 0.5

0.5 1.0

0.5 2.0

0.5 3.0

No. of shoots/ culture

7.89 + 0.39 9.9 + 0.76

13.42 + 0.62 17.8 + 0.38

SS + C SS + C

9.2 + 0.65 9.64 + 0.7 16.3 + 0.7 20.4 + 0.7 24.4 + 0.5 24.3 + 0.43

13.2 + 0.72 +C

9.2 + 0.42 10.2 + 0.76 2 1.8 + 0.82 21.2 + 0.61

Effect of BA

Effective multiple shoot induction from nodal explants was obtained on

MS medium supplemented with BA at different concentrations (0.5-3.0 mg/l).

The simulating effect of BA in the bud break and multiple shoot production was

highest (201 explant) at an optimal concentration (2mg/l). The percentage of

multiple shoot induction was declined with the increase in BA concentration

beyond the optimal level of 2mg/l .

Effect of Kn

Kinetin at varied concentrations (0.5 - 2.0 mg/l) on MS medium failed to

produce multiple shoots from nodal explants. However, single shoot formation

was observed at higher concentrations of Kn(2 1.0 mg/l) (Plate 12 A).

Effect of NAA + BA

Nodal explants showed highest shoot multiplication on MS medium

supplemented with a combination of NAA+BA combinations. The combination

also promoted shoot elongation. NAA (0.5 mg/l) and BA (3.0 mg/l) produced

maximum number of shoots (24) per nodal explant within 20-25 days (Plate 12

B). Increase or decrease of NAA or BA concentrations from the optimal levels

adversely affected shoot multiplication.

Effect of IAA + BA I

Murashige and Skoog's medium with combination of IAA (0.5 mg/l) and

BA (0.5 - 3.0 mg/l) was also effective in inducing multiple shoots from nodal

explants. However, the combination was less effective when compared to NAA

+ BA combination. At the optimal concentrations of IAA (0.5 mg/l) and BA

(2.0 mg/l) the nodal explants produced an average of 22 shoots. (Table 21)

iii) Direct shoot regeneration from leaf explants

Different levels of cytokinins (BA and Kn ) either singly or in combination

were tried to establish direct shoot regeneration from leaf explants. Among

these, MS medium fortified with BA (0.1-3mg/l) and BA (0.1-3mg/l) + Kn (0.1-

3mg/l) was effective for direct multiple shoot regeneration from leaf explants.

Direct shoot formation from the explant without any callusing was

obtained only on MS medium supplemented with 0. l mg/l BA. About 40% of

the leaf explants produced single shoots on the above medium(Plate 13 A,B)

(Table 22). At higher concentrations (>0.5mg/l) of BA multiple shoots were

formed with varying frequency . However, along with direct adventitious shoot

formation little amount of callusing was also observed on leaf explants.

Murashige and Skoog's medium containing BA (0.1 - 3.0mg/l) and Kn

(0.1 - 3.0mg/l) was most effective combination for direct shoot regeneration.

Optimal concentration of BA (2.0mg/l) and Kn (2.0mg/l) on MS medium

Table 22. Effect of growth regulators on shoot induction from leaf explants on N. alata

Data from 20 replicates in two experiments (Mean &SE) Growth period 50 days C = callusing

% of Response

30

40

45

65

65

50

60

7 5

75 70

Growth Regulators

(mdl) BA

0.1

0.5

1 .O

2.0

3 .O

BA + K n 0.1 0.1

0.5 0.5

1.0 1.0

2.0 2.0

3.0 3.0

No. of shoots/ culture

1.1 50.15

3.25 2 0.52 +C 11.620.27 + C

19.1420.61 + C

18.2 2 0.17 +C

2.3 1 2 0.24 4.2 2 12 +C

13.6 2 0.27 +C 27.1 20.41 + C

24.3 2 0.31 + c

Shoot length

3.48 0.26 '

3.4 + 0.3 1 3.14 2 0.18 2.98 2 0.27 2.92 5 0.62

3.7 + 0.28 3.32 2 0.47 3.14 2 0.65 2.96 2 0.14 2.87 2 0.23

enhanced proliferation and induced highest number (27fleaf) of

shoots from leaf explants (Plate 13 C).

iv) Effect of nutrient media

To select most suitable medium for shoot multiplication of N.

alata shoot tips were cultured on various media (MS, B5 and

White's) supplemented with 0.5mg/l NAA and 3mg/l BA. MS medium

was found as the most effective one on which highest number of

shoots (27) were developed followed by B5 (24) and White's (3)

media (Figure 5).

iv) Effect of sucrose concentration

Direct shoot proliferation and growth were greatly influenced by

the level of sucrose added in the culture medium. To find out the

optimal level of sucrose, shoot tip explants were cultured on MS

medium containing varying concentrations of sucrose (l-5%) along

with 0.5 mg/l NAA + 3.0 mg/l BA. Sucrose at 3% gave significant

result compared to other concentrations (Figure 6).

INDIRECT SHOOT REGENERATION

For the establishment of indirect shoot regeneration of N.

alata, various explants viz., leaves, internodes, petals, and roots

were cultured on MS medium fortified with different growth

regulators. When the explants were cultured on MS medium

supplemented with auxin only callusing was observed. However,

Figure 5. Ef fro

:ect of nutrient media on shoot induction m shoot tip explants on N. alata

BS+0.5NAA+3BA

Nutrient media

Figure 6. Effect of sucrose on shoot induction from shoot tips of N. alata

( Cultured on MS with 0.5mgll NAA +3mgll BA )

3 4

Sucrose %

when explants were cultured on MS medium supplemented with combination

of BA and an auxin, ( IAA or NAA) induction as well as regeneration of

shoots from calli were observed (Table 23).

Effect of NAA + BA

Different explants when cultured on MS medium supplemented with NAA

(0.5 mg/l) + BA (0.5 - 4.0 mg/l) induction of calli and regeneration of shoots

occurred on the same medium (Plate 17 D). In the case of root explants,

combination containing at least 1.0 mg/l BA was necessary for regeneration of

shoots from the calli. The calli induced on this combination were green and

friable with nodular structures (Plate 15 B) and regeneration was observed

within 30 days (Plate 17 C). 0.5 mg/l NAA + 3.0 mg/l BA in MS medium was

the optimal concentrations for indirect shoot regeneration for most of the

explants(P1ates 14 C; 15 D; 16 E). Among the different explants tested leaf

explants produced highest number of shoots (11) in the optimal combination

(Table 23).

Effect of IAA + BA

MS medium supplemented with IAA (0.5 - 1.0 mg/l) + BA (0.5 - 4.0

mg/l) was also effective for indirect shoot regeneration from various explants of

N. alata. But root explants required a higher concentration of BA (11.0 mg/l)

Table 23. Combined effect of auxins&cytokinins on callus induction and regeneration from internode, leaf,root & petal explants of N. alata

Data fkom 20 replicates in two experiments (Mean *SE) Growth period 50 days C= Callus

Petal

2.9 5 0.61

3.6 2 0.14

6.9 + 0.22 7.3 2 0.66

7.3 5 0.66

C only

2.1 2 0.7 1

3.9 2 0.18

6.5 2 0.28

7.1 2 0.33

7.2 2 0.41

1.8 2 0.34

C only

C only

Root

C Only

2.3 + 0.23 4.9 & 0.49

8.220.17

5.4 50.11

C only

C only

2.4 5 0.32

5.2 2 0.19

5.2 2 0.14

5.1 2 0.27

1.7 5 0.36

C only

C only

Leaf

3.8 t 0.37 4.6 50.46

9.1 t 0.22 11.22 0.19

11.1214.5

C Only

3.2 5 0.72

4.1 2 0.39

6.5 2 0.64

8.5 2 0.55

8.2 2 0.12

2.220.11

C only

C only

Growth Regulators (mgll)

NAA BA

0.5 0.5

0.5 1 .O

0.5 2.0

0.5 3 .O

0.5 4.0

1.0 0.5

IAA BA

0.5 1 .O

0.5 2 .O

0.5 3 .O

0.5 4.0

0.5 5 .O

1 .O 0.5

NAA Kn

0.5 3 .O

IAA Kn

0.5 3 .O

Internode

3.2 2 0.12

4.23 5 0.67

8.33 lf: 0.49

10.850.14

10.5t0.29

C Only

3.4 2 0.24

4.2 2 0.13

6.7 2 0.38

8.3 2 0.33

8.29 2 0.41

2.120.22

C Only

C only

for regeneration of shoots. Leaf explants showed best response for

regeneration in comparison with other explants (Table 23).

Callusing was established within 8-10 days (Plate 15 A) and shoot

multiplication occurred only after one month. The calli were white and friable

initially (Plate 14 B) later it turned green (Plate 16 B) and meristematic and

shoots were emerged out (Plate 16 D). The best response and higher

percentage of shoot multiplication was obtained on the optimal combination of

IAA (O.smg/l) and (4.0 mg/l)

Effect of NAA + Kn

Combinations of NAA (0.5 mg/l ) + Kn ( 0.5-4mg/1 ) in MS medium also

induced calli from all explants tested. The calli were pale white and friable but

no shoot formation occurred in the combination (Plate 16 A)(Table 23).

Effect of IAA + Kn

Murashige and Skoog's medium with IAA + Kn combinations induced

calli from all the explants (Table 23). However, the calli did not regenerate

shoots on the same medium. The calli obtained were pale white and friable

(Plate 17 A,B).

Effect of NAA

Murashige and Skoog's medium with NAA at the range 0.5 - 3.0 mg/l

effectively induced callus within 12 days from explants of shoot system (leaf,

petal, internode) and within 25 days from root explants. The calli were pale

white and friable (Plate 16 C). 2.0 mg/l was the optimum concentration of NAA

for induction and proliferation of calli from various explants of N. alata. Root

formation was also observed from the calli (Table 24).

Effect of IAA

Murashige and Skoog's medium with IAA at the range of 0.5 - 4.0 mg/l

induced callusing from various explants of N. alata within 20 days from leaf,

internode and petal explants. However, callus induction was delayed on root

explants for 10 more days. The calli were white and friable. Optimum

concentration of IAA for callus induction was 3.0 mg/l for all explants cultured

(Plate 14 A)(Table 24).

Effect of 2,4-D

I t was found that 2,4-D was the most effective auxin for callus induction

from various explants of N. alata. Calli were induced within 10 days on all the

explants tested (Table 24). Callus induction and proliferation were progressively

increased on 2,4-D concentrations and found 2.0 mg/l 2,4-D was the optimal

concentration for callus induction. The calli were pale yellow and friable (Plate

15C).

The calli obtained from various explants on MS medium supplemented

with auxin or auxin + kinetin were tested for their regenerative potential by

Table 24. Effect of auxins (NAA, LAA & 2,4-D) on callus induction from various explants

Growth Regulators

(mg/l)

NAA

0.5

1.0

2.0

3 .o IAA

0.5

1 .o 2.0

3 .o 4.0

2,4-D

0.1

0.5

1 .o 2.0

3 .o + =Very slight; Data fiom 20 replicates in two experiments Growth period 50 days

of N. alata Internode

+ & R

++&R

+++ & R ++&R

+ +

+++ U +

+

+ U

U

*+ +++

++ =Little; +++

Leaf l

+ + + & R

+++&R

+++&R ,

+ + ++ +++ +

+ ++ ++ tt++

+++ =Moderate ; +-H-+=

Root

- -

++&R

++&R

+ ++ ++ +

+ + ++ ++

Prohse

Petal

+ + ++ ++

+ + ++ ++ +

+ ++ ++ +++

t+t

subculturing on MS medium with 0.5 mg/l NAA + 3mg/l BA. All the calli

regenerated shoots when cultured on the above combination.

SOMATIC EMBRYOGENESIS

Somatic embryos of N. alata were formed within 60 days upon

subculturing of the embryogenic calli obtained from various explants on MS

basal medium. Direct somatic embryogenesis and subsequent plant

regeneration were also established from leaf explants.

i) Direct somatic em bryogenesis

Direct somatic embryogenesis was obtained from leaf explants of N.

alata on MS medium supplemented with 0.1 mg/l 2,4-D (Plate 18 A, B). The

mode of placing the explant on the nutrient medium showed marked influence

on the embryogenic response of the explant. When the adaxial side of the leaf

was touching the medium only direct embryoids were formed on the other side

(Plate 18 B). But when abaxial side was touching the medium, direct somatic

embryos and embryogenic calli were formed on the other side (Plate 18 A). At

higher concentrations of'2,4-D (2 0.5 mg/l) only embryogenic calli were formed

instead of direct somatic embryos (Table 25).

ii) Indirect somatic embryogenesis

Indirect somatic embryogenesis of N. alab was established by

subculturing embryogenic calli on MS basal medium

Table 25. Effect of 2,4-D IAA, NAA, BA & Kn on sohatic embryogenesis from leaf explants of N. alata

Data from 20 replicates in two experiments (Mean S E ) Growth period 50 days EC-Embryogenic calli NC-Non embryogenic calli

Growth Regulators (mgll) 2,4-D

0.1 I i I 0.5 l l

1 .O

l l

2.0

i 3.0 l I

I NAA

l I 0.1 1 I 0.5

i l 1 .O i 2.0 1 j 2,4-D+ Kn l l I 0.5 0.5 I 0.5 1.0 i I 2,4-D+ BA i o.5 0.5 1 0.5 1.0

Response

Direct embryos and Embryogenic

Calli EC

EC EC

EC

NC NC

NC

NC

NC

NC

NC NC

% of Response

80

8 5

8 5

8 0 70

70

65

70

75

40

70

60

65

No. of embryos foundlfrorn cali

4.3 5 0.27

6.7 2 0.33 10.2 + 0.62 8.2 5 0.24 8.4 2 0.22

- - - -

- -

-

a. Induction of embryogenic calli

Different explants of N. ahta were cultured on MS medium

supplemented with various growth regulators for the induction of embryogenic

calli.

Effect of growth regulators

Leaf explants were cultured on MS medium supplemented with various

growth regulators for the induction of embryogenic calli. The type of the auxin

and its concentration used in the medium significantly influenced the formation

of embryogenic callus (Table 25). Among the various combinations and

concentrations of growth regulators tested (2,4-D, NAA, 2,4-D+Kn and 2,4-

D+BA), MS medium supplemented with 2,4-D (0.1-3.0 mg/l) only produced

embryogenic calli from the explants (Table 25). The calli obtained were glossy

and pale white (Plate 18 C) initially, which later turned yellow. (The

embryogenic potential was later recorded by observing response of the calli on

MS basal medium in 60 days). Addition of BA or Kn along with 2,4-D favoured

callus proliferation. However, the calli were non embryogenic.

Effect of various explants

Among different explants cultured (leaf, internode, root and petal) on MS

medium supplemented with 1.0 mg/l 2,4-D, leaf explants produced calli with

highest embryogenic potential (the embryogenic potential was later recorded by

observing response of calli on MS hormone free medium) (Figure 7).

Effect of sucrose

The level of sucrose also had significant effect in inducing embryogenic

calli. Among the different concentrations of sucrose (2-6%) tested in MS

medium supplemented with 1.0 mg/l 2,4-D, medium containing 5% sucrose

produced calli with highest embryogenic potential (Figure 8).

b) Induction and maturation of somatic embryos

Somatic embryos were formed when embryogenic calli were cultured on

MS hormone free semi-solid (Plate 18 D) or liquid medium (Plate 18 E,F,G)

within 60 days. Liquid medium was superior to semi-solid medium for the

induction of somatic embryos from embryogenic calli. The embryogenic calli

obtained from leaf explants on MS medium supplemented with 1.0 mg/l 2,4-D

produced an average of 10 somatic embryos /g calli in liquid medium whereas

only 8 embryos were formed/g calli when cultured on semi-solid medium.

In general, the embryos passed through the normal developmental

stages (globular, heart, torpedo) (Plate 18 E) and underwent germination (Plate

18 H) when cultured on MS basal semi- solid medium with a frequency of over

70%. The plantlets derived through somatic embryogenesis were similar to

normal field grown plants.

Figure 7. Effect of various explants on somatic embryogenesis of N. alata

cultured on MS + I mgll2,4-D

l : Leaf Internode root Petal

Various explants

Figure 8. Effect of sucrose concentration on somatic embryogenesis of N. alata (Cultured on

MS + l mgII 2,4-D)

2 3 4 5

% of Sucrose tested

SYNSEEDS

Three percent sodium alginate and 50 mM CaCI2(2Hz0) solutions were

found to be the best for the encapsulation and subsequent germination

/development of somatic embryos/shoot buds of N. slab (Plate 19 A, B).

Encapsulated embryos were germinated within 30 days on MS basal

medium at a frequency of 60%. Encapsulated shoot buds did not show any sign

of growth on MS basal media even after 20 days of transfer. #However, about

70% encapsulated shoot buds showed signs of development within 15 days

when cultured on MS medium supplemented with 1.0 mg/l Kn. Storage at 4°C

for 6 months did not affect the gerrnination/development (Plate 19 C) of the

encapsulated propagules.

ROOTING I N VITRO

For root induction individual shoots (> 3cm) were excised and cultured

on half or full strength MS medium supplemented with various growth

regulators. Half strength MS medium was superior to full strength medium for

root induction of in vitro raised shoots of N. alata. Among the three auxins

(NAA, IAA and IBA) tested NAA was superior to other two for rooting of N.alata

shoots (Table 26) (Plates l 1 C;12 C 13 D;14 D; 15 E; 16 F; 17 E;19 D; 20 A).

Optimal concentration (0.5mg/l) of NAA on half strength MS medium induced

highest number of roots from in vifro raised shoots (Plate 20 A).

Table 26. Effect of ?4 and fup strength MS media & auxins (M, NAA & 2,4-D) on in vitro rooting of N. alata

Data from 20 replicates in two experiments (Mean &SE) Growth period 50 days

Root length

1.6 + 0.71 1.9 + 0.8

3.4 + 0.12 4.2 + 0.62 4.1 + 0.11 3.8 + 0.23

2.2 + 0.14 2.6 + 0.27 3.1 + 0.32

-

3.7 + 0.8 3.9 + 0.14

4.0 + 0.14

3.7 + 0.22

3.4 5 0.26

Growth Regulators mg/l

NAA

0.1

0.2

0.3

0.4

0.5

1 .O

IBA

0.1

0.5

1 .O

2.0

IAA

0.1

0.5

1 .O

MS full strength NAA 0.5

IBA 1 .O

IAA 1 .O

% of Response

40

50

70

85

90

90

- 70

80

85

- 70

70

8 5

70

80

No. of roots

3.12 + 0.24 4.3 + 0.18 8.7 + 0.36 9.2 + 0. l l 13.1 + 0.21 1 1.7 t 0.24

4.2 + 0.3 8.7 + 0.5 1 7.3 + 0.49

- 2.4 + 0.43 5.6 5 0.62

9.5 + 0.22

4.9 + 0.46

6.7 + 0.26

Table 26. Effect of % and fup strength MS media & auxins (IAA, NAA & 2,4-D) on in vitro rooting of N. alata

Data fiom 20 replicates in two experiments (Mean &SE) Growth period 50 days

Root length

1.6 0.71

1.9 2 0.8 3.4 + 0.12 4.2 + 0.62 4.1 iO.11 3.8 2 0.23

2.2 5 0.14 2.6 5 0.27 3.1 + 0.32

- 3.7 2 0.8 3.9 5 0.14

4.0 t 0.14

3.7 2 0.22

3.4 5 0.26

- Growth Regulators

mg/l NAA 0.1

0.2

0.3

0.4

0.5

1 .O

IBA 0.1

0.5

1 .O

2.0

IAA 0.1 0.5

1 .O

MS full strength NAA

0.5

IBA 1 .O

IAA 1 .O

% of Response

40

50

70

85.

90 90

- 70

80

85

- 70

70

8 5

70

80

No. of roots

3.12 2 0.24 4.3 2 0.18 8.7 2 0.36 9.2 + 0.1 l 13.1 t 0.21 11.7 + 0.24

4.2 2 0.3 8.7 2 0.5 1 7.3 2 0.49

- 2.4 + 0.43 5.6 0.62

9.5 + 0.22

4.9 + 0.46

6.7 2 0.26

Optimal concentration (0.5mgJl) of NAA on half strength MS medium induced

highest number of roots from in vitro raised shoots (Plate 20 A).

SECONDARY METABOLITE ANALYSIS

In the present studies Thin layer chromatography (TLC) was performed

to compare the alkaloids present in the roots of in vitroand field grown plants.

Root extracts from in vitro plants showed 4 compounds (spots) with Rf

values 0.031, 0.056, 0.0462 and 0.975. However, root extracts from in vivo

plants showed only 3 compounds (spots) with Rf values 0.031, 0.56 and 0.975.

This indicates the presence of an additional compound in li7 vitro roots

compared to in vivo roots (Plate 2 l )

ACCLIMATISATION AND TRANSFER TO FIELD CONDITIONS

Plantlets derived from different cultures with sufficient root and shoot

system were successfully hardened under laboratory conditions. The plantlets

were transferred after washing with sterilized distilled water into small plastic

pots containing sterile sand and soil in 1:1 ratio and were covered with

polythene bags or glass bottles ( Plate 20 B) for acclimatization. After 20-25

days they were eventually established in natural conditions (Plates 1lD; 12 D;

13 E; 14 E, 17 F; 18 I; 19 E). These plantlets were successfully transferred to

large pots/field showed high percentage of ( 85%) survival (Plate 20 C).

BIOCHEMICAL STUDIES

Biochemical analysis of primary calli (induced on MS medium supplemented

with IAA 2mg/l) and regenerating calli (on MS medium supplemented with IAA

0.5 mgfl + BA 2 mg/l) showed that there were marked differences between the

two calli in terms of biochemical characteristics. Metabolites like sugars,

proteins and phenolics were found to be high in the regenerating callus than in

the primary callus of N. alata. Peroxidase activity was also high in the

regenerating callus than in the primary callus (Table 27). Polypeptide pattern

analysis of the two calli showed more types of polypeptides in the regenerating

callus then in the primary callus (Figure 9 ).

Table 27. Changes in metabolite levels and activity of peroxidase in prihary and regenerating callus cultures of N. data.

Data represents an average of4 replicates scored in 50 clays old callus.

Type of Cdus

Regenmating callus Primary callus

Figure 9.

,SDS Page of NavganuL alatal

M-Molecular marker R-Regeamting callus P-Primary callus

Total sugar %

1 8.3

10.6

Total phmiics mg/g;Fr. Wt, 3.9

3.6

Total prdein YO

0.44

0.34

P m a r i b Unitsl mg Protein 50

12

PLATE 11. Micropropagation of slatafrom shoot tip.

A. Multiple shoots induced on MS+NAA (0.5 mg/l) +BA (3mg/l)

B. Multiple shoot showing elongation.

C. Root induction on 1/2 MS+NAA (0.2mgIl)

D. Hardened plantlet in small pot.

PLATE 12. Micropropagation of N. alatafrom nodes.

A. Shoot induction on MS +Kn (2mg/l)

B. Multiple shoots induced on MS +NAA (O.Smg/l) + BA (3mg/l) C. Roots induced on V2 MS+NAA (0.5mg/l)

D. Hardened plantlet in small pot.

'S- -

PLATE 13. Micropropagation of N. a/atafrom leaf explant.

A. Shoot induction on MS +BA (O.lmg/l) ( l 0 days old)

B. Shoot induction on MS +BA (O.lmg/l) (15 days old)

C. Multiple shoots induced on leaf on MS +BA (2mg/l)+Kn

(2mgII)

D. Rmts induced on 1/2 MS + IAA (2mg/l) E. Hardened Plantlet in small pot.

PLATE 14. Micropropagation through indirect shoot regeneration of

H.keraIensis from internodes.

A. Callus induced on MS+IAA (0.5mg/l)

B. Callus induced on MA+ NAA (0.5mg/l)+BA (lmg/l)

C. Callus regeneration on MS +NAA (0.5mg/l) +BA (4mg/l)

D. Roots induced on '/2 MS +IAA (0.5mg/l)

E, Hardened plantlet in small pot.

I= - PLATE 14

PtATE H. Indirett otgsnogenesis from leaves of N. a/afa

A. Callus induced on MS+ IAA (0.5mg/l) + BA (2mg/l) B. Callus induced on MS+NAA (0.5mg/l)+ BA (3mg/l)

C. Callus induced on MS +2,4-D (2mg/l)

D. Shoot regeneration from callus on MS+ NAA (0.5mg/l)+BA

(3mg/l) (50 days old)

E. Roots induced on l/2 MS+ IAA (0.5mgll)

, \ L I l,:. . ' .

I PLATE IS

PLATE 16. Indirect organogenesis from petals of N. a/ata.

l

A. Callus induced on MS + IAA (0.5 mgjl). B. Callus induced on MS + IAA (0.5mg/l)+ BA (1mgjL) (20 days

old)

C. Callus induced on MS + NAA (0.5 mgjl) + BA (lmgjl) ( 20 days)

D. Indirect shoot regeneration on MS + IAA (0.5 mgjl) + BA (2mgIl) ( 40 days 1.

E. Rooting on MS + IBA ( l mg/l)

PLATE l L

PLATE 17. Microproparation through indirect shoot regeneration

from roots of N. alata.

A. Callus induced on MS + IAA ( lmg/l) ( l 0 days). B. Callus induced on MS + IAA ( lmg/l) (25 days). C. Indirect shoot regeneration on MS + NAA (0.5 mg/l) + BA (1

m9/1)

D. Indirect shoot regeneration on MS + NAA (0.5mg/l) + BA ( 4 mg/l)( 40 days

E. Roots induced on l/z MS + NAA ( 0.1 mg/l) F. Hardened plantlet obtained through indirect shoot

regeneration in small pot.

PLATE 18. Plant regeneration via. Somatic embryogenesis of N. alats,

A. Induction of direct somatic embryos and embryogeneic callus

from leaf on MS + 2,4-D (0.1 mg/l) (abaxial side in contact with the medium).

B. Induction of direct globular somatic embryos from leaf on MS

+ 2,4-D (0.1 mg/l) ( adaxial side in contact with medium) C. Embryogenic callus induced from internode on MS + 2,4-D (

1 mgll)

D. Globular embryos developed from embryogenic callus on MS

basal medium.

E. Somatic embryos showing different stages of development.

F. Globular somatic embryos formed in suspension culture.

G. Heart shaped somatic embryos.

H. Embryo germinated plantlet.

I. Hardened plantlet in small pot.

PLATE 18

PLATE 19. Encapsulation and germination of somatic embryos/

shoot buds of N. alata.

A. Encapsulated shoot buds.

B. Encapsulated somatic embryos

C. Germinating synseed.

D. Plantlet developed from synseed.

E. Hardened plantlet in small pot.

PLATE 19

PLATE 20. Steps in hardening and field transfer of N. data.

A. Plantlet with well developed shoot and root system.

B. Plantlet under hardening

C. Hardened plant under field conditions in pot.

L PLATE 20

PLATE 21. TLC analysis (for secondary metabolites) of roots from

h vifro and field grown plants.

PLATE. 21